The p.N478D missense mutation in human mitochondrial poly(A) polymerase (mtPAP) has previously been implicated in a form of spastic ataxia with optic atrophy. In this study, we have investigated fibroblast cell lines established from family members. The homozygous mutation resulted in the loss of polyadenylation of all mitochondrial transcripts assessed; however, oligoadenylation was retained. Interestingly, this had differential effects on transcript stability that were dependent on the particular species of transcript. These changes were accompanied by a severe loss of oxidative phosphorylation complexes I and IV, and perturbation of de novo mitochondrial protein synthesis. Decreases in transcript polyadenylation and in respiratory chain complexes were effectively rescued by overexpression of wild-type mtPAP. Both mutated and wild-type mtPAP localized to the mitochondrial RNA-processing granules thereby eliminating mislocalization as a cause of defective polyadenylation. In vitro polyadenylation assays revealed severely compromised activity by the mutated protein, which generated only short oligo(A) extensions on RNA substrates, irrespective of RNA secondary structure. The addition of LRPPRC/SLIRP, a mitochondrial RNA-binding complex, enhanced activity of the wild-type mtPAP resulting in increased overall tail length. The LRPPRC/SLIRP effect although present was less marked with mutated mtPAP, independent of RNA secondary structure. We conclude that (i) the polymerase activity of mtPAP can be modulated by the presence of LRPPRC/SLIRP, (ii) N478D mtPAP mutation decreases polymerase activity and (iii) the alteration in poly(A) length is sufficient to cause dysregulation of post-transcriptional expression and the pathogenic lack of respiratory chain complexes.

The French-Canadian variant of COX-deficient Leigh syndrome (LSFC) is unique to Québec and caused by a founder mutation in the LRPPRC gene. Using whole exome sequencing, Oláhová et al. identify mutations in this gene associated with multisystem mitochondrial disease and early-onset neurodevelopmental problems in ten patients from different ethnic backgrounds.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic neoplasm resulting from the malignant transformation of T-cell progenitors, and comprises approximately 15% and 25% of pediatric and adult ALL cases respectively. It is well-established that activating NOTCH1 mutations are the major genetic lesions driving T-ALL in most patients, but efforts to develop targeted therapies against this pathway have produced limited success in decreasing leukemic burden and come with significant clinical side effects. A finer detailed understanding of the genetic and molecular mechanisms underlying T-ALL is required identify patients at increased risk for treatment failure and the development of precision medicine strategies. Generation of genetic models that more accurately reflect the normal developmental history of T-ALL are necessary to identify new avenues for treatment. The DNA methyltransferase enzyme DNMT3A is also recurrently mutated in T-ALL patients, and we show here that inactivation of Dnmt3a combined with Notch1 gain-of-function leads to an aggressive T-ALL in mouse models. Moreover, conditional inactivation of Dnmt3a in mouse hematopoietic cells leads to an accumulation of immature progenitors in the thymus which are less apoptotic. These data demonstrate that Dnmt3a is required for normal T-cell development, and acts as a T-ALL tumor suppressor.

SUMMARY How specific genetic lesions contribute to transformation of non-malignant myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS) to secondary acute myeloid leukemia (sAML) are poorly understood. JARID2 is lost by chromosomal deletions in a proportion of MPN/MDS cases that progress to sAML. In this study, genetic mouse models and patient-derived xenografts demonstrated that JARID2 acts as a tumor suppressor in chronic myeloid disorders. Genetic deletion of Jarid2 either reduced overall survival of animals with MPN or drove transformation to sAML, depending on the timing and context of co-operating mutations. Mechanistically, JARID2 recruits PRC2 to epigenetically repress self-renewal pathways in hematopoietic progenitor cells. These studies establish JARID2 as a bona fide hematopoietic tumor suppressor and highlight potential therapeutic targets.

Compounds that impair the synthesis of either mitochondrial DNA (mtNDA) or mtDNA-encoded proteins reduce the levels of 13 proteins essential for oxidative phosphorylation, leading to a decrease in mitochondrial ATP production. Toxicity caused by these compounds is seldom identified in 24 to 72 hr cytotoxicity assays due to the low turnover rates of both mtDNA and mtDNA-encoded proteins. Here, we describe three high-throughput screening assays that detect compounds that affect mtDNA-encoded protein levels. All three assays measure the levels of two proteins, one a mtDNA-encoded protein synthesized on mitochondrial ribosomes and the other, a nuclear DNA-encoded protein synthesized on cytosolic ribosomes. The first assay measures the levels of these two proteins by quantitative image analysis and requires a high-content imaging system. The second assay is an in-cell immunoassay that utilizes infrared dyes for detection of the two proteins and, thus, requires a LI-COR Odyssey system. The third assay is an in-cell immunoassay that utilizes colorimetric detection of the two proteins and requires an absorbance microplate reader.

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Genome sequencing efforts have identified virtually all of the important mutations in adult myeloid malignancies. More recently, population studies have identified cancer-associated variants in the blood of otherwise healthy individuals as they age, a phenomenon termed clonal hematopoiesis of indeterminate potential (CHIP). This suggests that these mutations may occur in hematopoietic stem cells (HSCs) long before any clinical presentation but are not necessarily harbingers of transformation because only a fraction of individuals with CHIP develop hematopoietic pathologies. Delineation between CHIP variants that predispose for disease versus those that are more benign could be used as a prognostic factor to identify individuals at greater risk for transformation. To achieve this, the biological impact of CHIP variants on HSC function must be validated. One variant that has been identified recurrently in CHIP is a gain-of-function missense mutation in the imprinted gene GNAS (Guanine Nucleotide Binding Protein, Alpha Stimulating). In this study, we examined the effect of the GNAS variant on HSC function. Ectopic expression of GNAS supported transplantable HSC activity and improved lymphoid output in secondary recipients. Because declining lymphoid output is a hallmark of aging, GNAS mutations may sustain lymphoid-biased HSCs over time and maintain them in a developmental state favorable for transformation.